μ-opioid agonist-induced activation of G-protein-coupled inwardly rectifying potassium current in rat periaqueductal gray neurons

Abstract

The characteristics of the inwardly rectifying K + current activated by a μ-type opioid agonist, d-Ala 2,N-MePhe 4,Gly 5-ol-enkephalin (DAMGO), were examined in the acutely dissociated rat periaqueductal gray neurons using the nystatin-perforated and the conventional whole-cell recording modes under voltage-clamp conditions. DAMGO activated inward currents in a concentration- and voltage-dependent manner. The DAMGO-induced current was an inwardly rectifying K + current ( I DAMGO) which was sensitive to K + channel blockers, quinine and Ba 2+ but insensitive to Cs + and tetraethylammonium. In the conventional whole-cell clamp mode, guanosine 5′- O-(2-thiodiphosphate) trilithium salt (GDP βs, 0.4 mM) inhibited the amplitude of I DAMGO to 28% of that of the initial current. After the intracellular perfusion with guanosine 5′-[loc]O-(3-thiotriphosphate) tetralithium salt (GTP γs, 0.4 mM) for 1 min, the first application of DAMGO irreversibly activated I DAMGO. By the extracellular application of N-ethylmaleimide at a concentration of 50 μM for 2 min, I DAMGO was completely abolished. When a conventional whole-cell patch was made with a patch-pipette containing 1 μg/ml of pertussis toxin together with 1 mM of β-nicotinamide adenine dinucleotide, I DAMGO gradually declined to about 41% of its initial amplitude. The extracellular application of second messenger modulators including protein kinase inhibitor (staurosporin), protein kinase A activators (forskolin, 3-isobutyl-1-methyl-xanthine and dibutyryladenosine 3′5′-cyclic monophosphate) and protein kinase C activators (phorbol-12-myristate-13-acetate and 1-oleoyl-2-acetyl-sn-glycerol) had no effect on I DAMGO. These results suggest that (i) DAMGO-activated inwardly rectifying K + current is mediated by pertussis toxin-sensitive guanine nucleotide binding proteins (G-proteins); (ii) the types of G protein involved in I DAMGO are G i and/or G o; and (iii) the G-proteins exert their roles in I DAMGO without any mediation of the second messenger systems.